Basil seedling production environment influences subsequent yield and flavor compound concentration during greenhouse production

Radiation intensity and carbon dioxide (CO2) concentration can be precisely controlled to manipulate plant yield and quality. Due to increased plant densities during seedling production, fewer inputs per plant are required, creating the potential to increase production efficiency. Therefore, the objectives of this research were to: 1) quantify the extent radiation intensity and CO2 concentration under sole-source lighting influence morphology and yield of sweet basil (Ocimum basilicum) seedlings, and 2) determine if differences in morphology, yield, and volatile organic compound (VOC) concentration persist after transplant in a common environment. Sweet basil ‘Nufar’ seedlings were grown in growth chambers with target CO2 concentrations of 500 or 1,000 μmol·mol‒1 under light-emitting diodes (LEDs) providing target photosynthetic photon flux densities (PPFD) of 100, 200, 400, or 600 μmol·m‒2·s‒1 for 16 h per day. After two weeks, seedlings were transplanted into a common greenhouse environment and grown until harvest. At transplant and three weeks after transplant (harvest), growth and developmental differences were quantified along with key terpenoid and phenylpropanoid concentrations at harvest. Radiation intensity and CO2 interacted influencing many aspects of plant morphology, though CO2 concentration effects were less pronounced than those of radiation intensity. As radiation intensity during seedling production increased from 100 to 600 μmol·m‒2·s‒1, basil seedlings were 38% taller, had a 713% larger leaf area, and had 65% thicker stems; at harvest, plants were 24% taller, had 56% more branches, 28% more nodes, 22% thicker stems, and weighed 80% more when fresh and dry. Additionally, after growing in a common environment for three weeks, eugenol concentration was greater in plants grown under a PPFD of 600 μmol·m‒2·s‒1 as seedlings compared to lower intensities. Therefore, increasing radiation intensity during seedling production under sole-source lighting can carry over to increase subsequent yield and eugenol concentration during finished production.

Introduction increased anthocyanin, phenolic, and flavonoid concentrations. Chang et al. [21] reported that as the DLI delivered for two weeks to young seedlings increased from 5.3 to 24.9 mol�m -2 �d -1 , total VOC content of basil increased. In particular, the relative content of eugenol and linalool increased~300% and~400%, respectively, while methyl eugenol relative content decreased bỹ 80%. Carbon dioxide can also influence secondary metabolite production. For example, linalool, a compound of interest in basil, is also present in strawberry (Fragaria ananassa) [22]. An increase in CO 2 concentration from~350 to~950 μmol�mol -1 increased linalool concentration during strawberry production [22]. By investigating the individual and combined influences of DLI and CO 2 on VOC content and concentration, CE growers can work toward optimizing growing conditions that increase plant quality (VOC content and concentration) and yield (fresh mass).
Although technological advances have made indoor plant production more economically feasible, a better understanding of how to leverage environmental controls to improve crop productivity, quality, and energy efficiency is needed [23]. While researchers have been mainly focused on investigating the influence of environmental variables on finished-stage crops, the potential to improve high-density young plant production creates an opportunity to spread potentially greater input costs over a larger number of plants. Therefore, the objectives of this research were to: 1) quantify the extent radiation intensity and CO 2 concentration during seedling production influence yield, 2) determine if physiological and morphological differences remain present after transplant in a greenhouse, and 3) determine if differences in VOC concentration due to radiation intensity at the seedling stage remain present through harvest in a common greenhouse environment. Our hypotheses were that 1) growth would increase as radiation intensity and CO 2 increased, 2) there would be a positive interactive effect between CO 2 concentration and radiation intensity, where the effects of elevated CO 2 concentration would be more pronounced when the radiation intensity was higher, and 3) increased VOC concentrations at transplant would not persist through finishing in a common environment due to dilution during rapid growth.

Seedling production
Sweet basil 'Nufar' (Johnny's Selected Seeds, Fairfield, ME) was selected based on disease resistance and comparatively high yield results from Walters and Currey [24]. Seeds were sown two per cell in stone wool cubes (2.5 × 2.5 × 4 cm, AO plug; Grodan, Roermond, Netherlands) and 200-cell flats were placed in one of two walk-in growth chambers (Hotpack environmental room UWP 2614-3; SP Scientific, Warminster, PA) on 7 Aug. 2017, 10 Nov. 2017, and 22 Jan. 2018. Seeds and seedlings were grown and the environmental conditions were controlled and monitored as reported in Walters et al. [25].
Light-emitting diodes (LEDs) provided 20:40:40 blue:green:red radiation ratios (%), a red: far-red ratio of 13:1, and target radiation intensities of 100, 200, 400, or 600 μmol�m -2 �s -1 photosynthetic photon flux density (PPFD) for a 16-h photoperiod to create daily light integrals (DLIs) of 6, 12, 23, or 35 mol�m -2 �d -1 . Fixture density and hanging height were adjusted to achieve target radiation intensities. Radiation intensity and spectrum were measured at four corners and in the center of the seedling flat with a spectroradiometer (PS-200; StellarNet, Inc., Tampa, FL) to quantify the intensities and spectrum across the growing area (Fig 1,  Table 1). Target carbon dioxide concentrations of 500 and 1000 μmol�mol -1 were maintained by injecting compressed CO 2 to increase concentrations and scrub CO 2 using soda lime scrubber (Environmental Growth Chambers) to decrease concentrations. Concentrations were measured with a CO 2 sensor (GM86P; Vaisala, Helsinki, Finland) and logged by a C6 Controller (Environmental Growth Chambers) every 5 s (Table 1).

Finished plant production
Two weeks after sowing, 17 seedlings were transplanted into 0.9-m-wide by 1.8-m-long deepflow hydroponic systems (Active aqua premium high-rise flood table; Hydrofarm, Petaluma, CA) in a glass-glazed greenhouse. Baskets holding the seedlings were placed in 4-cm-diameter holes, 20-cm apart, in 4-cm thick extruded polystyrene foam floating on the nutrient solution. The nutrient solution consisted of reverse osmosis water supplemented with 12N-1.8P-13.4K water-soluble fertilizer (RO Hydro FeED; JR Peters, Inc.) and MgSO 4 providing twice the concentrations reported during seedling production. Electrical conductivity (EC) and pH were measured (HI991301 Portable Waterproof pH/EC/TDS Meter; Hanna Instruments, Woonsocket, RI) and adjusted to 1.56 mS�cm -1 and 6.0, respectively, by adding fertilizer, reverse osmosis water, potassium bicarbonate, or sulfuric acid. Air pumps (Active aqua 70 L�min -1 commercial air pump; Hydrofarm) and air stones (Active aqua air stone round 4"x1"; Hydrofarm) were used to increase dissolved oxygen concentrations.
The 16-h (0600 to 2200 HR) photoperiod consisted of natural photoperiods (lat. 43˚N) and day-extension lighting from high-pressure sodium (HPS) lamps providing a supplemental PPFD of~75 μmol�m -2 �s -1 to achieve target DLIs of 13 to 17 mol�m -2 �d -1 . The target average daily temperature was a constant 23˚C. Exhaust fans, evaporative-pad cooling, radiant steam heating, and supplemental lighting was controlled by an environmental control system (Integro 725; Priva North America, Vineland Station, ON, Canada). Shielded and aspirated 0.13-mm type E thermocouples (Omega Engineering) measured air temperature, infrared thermocouples (OS36-01-T-80F; Omega Engineering) measured leaf temperature, and quantum sensors (LI-190R Quantum Sensor; LI-COR Biosciences) placed at canopy height recorded PPFD. Every 15 s, a CR-1000 datalogger (Campbell Scientific) collected environmental data and hourly means were recorded (Table 2).

Growth, development, and VOC data collection and analysis
At transplant and three weeks after transplant (harvest), height from the substrate surface to the meristem, leaf area of two (seedling) or four (harvest) most recently fully expanded leaves Table 1. The date of sweet basil 'Nufar' (Ocimum basilicum) seed sowing, target and actual CO 2 concentration (± SD), target and actual radiation intensity (± SD), and average daily air, canopy, and substrate temperature (± SD) during the seedling growth stage (2 weeks).

Statistical design and analysis
The seedling portion of this experiment was organized the same as Walters et al. [25], a splitplot design with two CO 2 concentrations (two growth chambers) as the main factor and four radiation intensities as the sub factor. Finished greenhouse production was organized in a randomized complete block design with seedlings from the growth chamber blocked by treatment. The experiment was completed thrice over time for growth and development analysis (n = 30), and twice in time for GCMS analysis (reps 2 and 3; n = 20). Analysis of variance and t-tests were performed using JMP (version 12.0.1, SAS Institute Inc., Cary, NC). When interactions (CO 2 concentration × radiation intensity) were not significant, data were pooled (n = 60). Linear and quadratic regression analyses were conducted using Sigma Plot (version 11.0, Systat Software Inc., San Jose, CA).

Finished volatile organic compound concentrations
After seedlings were transplanted into a common greenhouse environment and grown for three weeks, there was no difference in linalool, 1,8 cineole, or methyl chavicol concentrations due to radiation intensity or CO 2 concentration provided during the seedling stage (Fig 4A, 4B  and 4D). However, as the radiation intensity during seedling production increased, an overall quadratic increase in eugenol concentration persisted through finishing (Fig 4C). There were minimal differences in eugenol concentration among plants grown under a PPFD of 100, 200, or 400 μmol�m -2 �s -1 during the seedling stage, three weeks after transplant. Increasing the radiation intensity to 600 μmol�m -2 �s -1 during the seedling stage increased eugenol concentration 44% to 183% (1,327 to 1,946 ng�mg -1 dry mass) compared to the lower radiation intensity treatments. There was no effect of CO 2 concentration on VOCs.

CO 2 concentration did not influence mass
Contrary to our hypothesis, CO 2 concentration did not influence fresh or dry mass at transplant or harvest (Figs 2B and 2D, 3E and 3F). While most research illustrates increased CO 2 concentrations can increase biomass, there are a few reasons plants may not respond to elevated CO 2 concentrations. Plants can become acclimated to elevated CO 2 concentrations, with prolonged exposure becoming inhibitory to photosynthesis; however, the extent and presence of acclimation or negative effects are species-and potentially, production stage-dependent [27,29]. For example, Sage et al. [27] reported that long-term elevated CO 2 (900 to 1000 μmol�mol -1 ) negatively affected the photosynthetic rate of C 3 plants such as kidney bean 'Linden' (Phaseolus vulgaris), eggplant (Solanum melongena), and cabbage (Brassica oleracea), but increased the photosynthetic rate of C 3 plant lambsquarters (Chenopodium album). While the main benefit of elevated CO 2 is the favoring of carboxylation activity over oxygenation activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), initial increased photosynthetic rates can cause excess carbohydrate production resulting in feedback inhibition, thus a reduction in photosynthesis [27][28][29]. Additionally, in some species such as cabbage, lambsquarters, petunia (Petunia ×hybrida), elevated CO 2 decreased Rubisco content and/or activity [27,29], induced stomatal closure, and/or decreased stomatal density [27,29,30].
The lack of CO 2 effect on biomass could also be due to the concentration being near or above the CO 2 saturation point. Previous research determined that increasing CO 2 concentrations from 360 to 620 μmol�mol -1 increased~4-week old basil (cultivar not reported) fresh mass 40% when grown under a radiation intensity of 150 μmol�m -2 �s -1 (8.6 mol�m -2 �d -1 ) [15]. We hypothesize that the increased mass with increased CO 2 concentration reported by Al Jaouni et al. [15] was due to CO 2 concentrations being below the CO 2 saturation point. Park et al. [31] determined the CO 2 saturation point of basil was 729 μmol�m -2 �s -1 when sweet basil (cultivar not reported) was acclimated to 20˚C, 400 μmol�mol -1 CO 2 , and a radiation intensity of 150 μmol�m -2 �s -1 (9.7 mol�m -2 �d -1 ). Additionally, as CO 2 concentration approaches the saturation point, increases in mass are attenuated. Since we utilized 500 and 1,000 μmol�mol -1 CO 2 , 500 μmol�mol -1 may have been too similar to the CO 2 saturation point and 1,000 μmol�mol -1 may have been above the saturation point, resulting in no discernable difference in mass. If we had maintained CO 2 concentrations below 500 μmol�mol -1 , differences may have occurred; however, this hypothesis would have to be tested.
Another contributing factor could be that CO 2 utilization may be limited by environmental factors including temperature. As temperature increases, Rubisco has a higher affinity for oxygen; therefore, the positive influence of elevated CO 2 on photosynthesis increases as temperature increases from approximately 20 to 35˚C, though the effect is species-dependent [32]. In this study, basil was grown at~23˚C. If temperatures had been higher and closer to the optimal temperature for sweet basil growth and development of 32 to 35˚C [33], the elevated CO 2 concentration may have been more likely to have had an effect on fresh mass.

CO 2 concentration influenced morphology
The increased height, leaf area, and stem width due to lower radiation intensities (100 to 400 μmol�m -2 �s -1 ; Fig 2A, 2C and 2E) with elevated CO 2 concentration were likely due to differing biomass partitioning, since neither fresh nor dry mass were affected. In wheat 'WW15' (Triticum aestivum), leaf area index increased at elevated CO 2 concentrations under lower radiation conditions [34]. However, there are conflicting reports on the effect of elevated CO 2 on leaf area. In separate experiments under different environmental conditions, leaf area of tomato 'Minibelle' increased as CO 2 concentration increased [35], however, tomato 'Findon Cross' leaf area was unaffected [36]. The lower height, leaf area, and stem width when basil seedlings were under a radiation intensity of 600 μmol�m -2 �s -1 and elevated CO 2 was counterintuitive (Fig 2A, 2C and 2E). It is well documented that as radiation intensity increases, leaf �s -1 ) for a 16-h photoperiod to create daily light integrals of (6, 12, 23, or 35 mol�m -2 �d -1 ) for two weeks and then transplanted in a common greenhouse environment and grown for three weeks. Each symbol represents the mean of 20 plants ± SE. Lines represent linear or quadratic regression. �� indicates significant at P � 0.01.
https://doi.org/10.1371/journal.pone.0273562.g004 thickness increases [8]. It could be possible that increased tissue thickness can impact plant responses to elevated CO 2 , however, additional morphological and physiological data is needed to confirm or reject the hypothesis.

Seedling production conditions influence basil yield and quality at harvest
Increasing radiation intensity from 100 to 600 μmol�m -2 �s -1 (5.8 to 34.6 mol�m -2 �d -1 ) increased seedling fresh mass 284% (Fig 2D), and an 80% increase in fresh mass yield persisted through finishing in a common environment (Fig 3C). Radiation intensity during propagation of floriculture crops can have a profound effect on subsequent growth and development. For example, increasing DLI from 4.1 to 14.2 mol�m -2 �d -1 during seedling production hastened flowering and reduced shoot dry weight at flower for celosia (Celosia argentea var. plumosa), impatiens (Impatiens walleriana), French marigold (Tagetes), and pansy (Viola) [37]. However, the reduction in dry mass can be primarily attributed to earlier flowering and thus, a shorter production duration. In the present study, plants were not grown until anthesis, but were harvested at the same time. Therefore, the influence of radiation intensity observed at transplant persisted, but was attenuated. Similar to the floriculture studies, development, including node and branch number, was hastened by increased radiation intensity during propagation in our study.
In addition to increased yield at harvest, and contrary to our hypothesis, the higher eugenol concentrations in seedlings grown under high radiation intensities [25] persisted at harvest (Fig 4C). Walters et al. [25] observed that increasing radiation intensity from 100 to 600 μmol�m -2 �s -1 (5.8 to 34.6 mol�m -2 �d -1 ) during basil seedling production increased 1,8 cineole, linalool, and eugenol concentrations. In a study investigating the influence of radiation quality on basil VOCs, researchers suggested that specific light treatments during germination and early seedling development "may install a particular developmental/metabolic pattern that influences potential to produce flavor and aroma compounds later" [38]. Our results suggest that the production or biosynthetic pathway of some compounds may be more sensitive to early environmental conditions than others. From a crop quality perspective, growers have indicated that their customers would pay more for crops with increased flavor [17]. However, consumer sensory panels have determined that when consumed raw and alone, there is an upper limit to consider and consumers do not always prefer basil with a more intense flavor [25]. Therefore, the benefit of an elevated eugenol concentration at harvest is not clear and may be situational.

Efficiency implications
In this study, we investigated the effect of increased inputs during the seedling stage to increase yield and secondary metabolite accumulation at harvest. By sowing seeds in a 200-cell tray (1,290 cm 2 per flat, 6.45 cm 2 per cell), the planting density is 1,550 plants per m 2 . Seedlings were transplanted 20-cm apart with a planting density of 25 plants per m 2 . Therefore, planting density was 62 times greater during propagation than finished (harvest) production. Additionally, in this study, the duration of seedling production was 2/3 that of finishing (two weeks compared to three weeks). Taking both the increased planting density and shorter production duration into account, the increase in lighting cost per plant could be discounted by 93 times during seedling versus finished production.
Therefore, in this case, the cost per plant of increasing the radiation intensity from 100 to 600 μmol�m -2 �s -1 during propagation was~5% that of the cost during finished production. Since the increase in yield at the finishing stage was 80% greater when seedlings were grown under 600 μmol�m -2 �s -1 compared to 100 μmol�m -2 �s -1 (34.6 compared 5.8 mol�m -2 �d -1 ), increasing radiation intensity during propagation increases subsequent yield and eugenol concentration while reducing costs.

Conclusions
Increasing radiation intensity from 100 to 600 μmol�m -2 �s -1 (5.8 to 34.6 mol�m -2 �d -1 ) during basil seedling production is an effective method of improving subsequent yields and increasing eugenol concentration. Although elevated CO 2 concentrations did not influence fresh or dry mass, future research is needed to determine at what stage of production elevated CO 2 concentrations could increase basil growth and secondary metabolite concentrations, if any. With these data, environmental controls, especially radiation intensity and CO 2 concentration, can be better leveraged during young plant production to improve crop productivity, quality, and energy efficiency not only at transplant, but also after finishing in a common environment. Additionally, this research can serve as a basis for scientific advances in dynamic environmental control.